Results of numerous studies point to an abnormality
of the immune function in autism,as
well as active, ongoing inflammation in the
GI tract, the brain and the cerebrospinal fluid (CSF).

A recent study by Vargas et al [15546155]
investigated the presence of immune activation in postmortem
brain specimens and CSF from subjects with autism. The authors
found active neuroinflammation in multiple areas of the brain,
for example in the cerebral cortex and white matter, and in
the cerebellum. A marked migroglial and astroglial activation
was also found, as well as the presence of an altered
cytokine pattern, with macrophage chemoattractant protein
(MCP)-1 and tumor growth factor-beta1 (TGF-beta1) being the
most prevalent cytokines. There was also an accumulation of
macrophages and monocytes, and a marked absence of lymphocytes
and antibodies, pointing toward an innate neuroimmune
activation with the absence of adaptive immune system/T cell
activation in the brain. In addition, an enhanced proinflammatory
cytokine profile was observed in the CSF, including once more
a marked increase in MCP-1. These observations resemble findings
in other neurological disorders in which elevations in cytokine
levels is associated with the pathogenesis of neuroinflammation,
neurotoxicity and neuronal injury and subsequent behavioural
and cognitive impairments, for example HIV-associated dementia
and multiple sclerosis [15288500,
11282546,
16875710,
9852582].
Another study examinig brain tissue of found that proinflammatory
cytokines TNF-alpha, IL-6 and GM-CSF, IFN-gamma and IL-8 were
significantly increased in the brains of ASD patients compared
with the controls. However the Th2 cytokines (IL-4, IL-5 and
IL-10) showed no significant difference. The Th1/Th2 ratio was
also significantly increased in ASD patients, suggesting localized
brain inflammation and autoimmune pathology [19157572].
Further findings suggestive of autoimmune pathogenesis in a
subgroup of patients revealed higher levels of antibodies directed
against human cerebellar protein extracts [18706993],
and high anti-nuclear antibody seropositivity in ASD patients
compared to controls. In addition, Anti-nuclear antibody seropositivity
had significant positive associations with disease severity,
mental retardation and electroencephalogram abnormalities [19135624].
Autistic children were found to have significantly higher serum
anti-myelin-associated glycoprotein antibodies than healthy
children [19073846].

Another
investigation into inflammatory markers in the brain tissue
of patients with autisms revealed significantly increased levels
of several proinflammatory cytokines (TNF-alpha, IL-6 and GM-CSF,
IFN-gamma, IL-8). The Th1/Th2 ratio was also significantly increased
in ASD patients, suggesting that localized brain inflammation
and autoimmune disorder may be involved in the pathogenesis
of ASD [19157572].

Of some
interest in this context may be the role of IL-6, a cytokine
that is known to modulate cellebral function and enhance neurotoxicity
through influencing Purkinje neuron physiology and electrical
activity. Animal experiments illustrate that, during early pre
and postnatal development, inflammatory cytokine challenge can
induce various psychological, behavioral and cognitive impairments
[17804539,
16147952,
9852582].
At the same time the expression of many cytokines, including
MCP-1, in neurons and glial cells seems to be upregulated by
increased intracellular calcium triggered by membrane depolarisation
[11102468,
10943723]
(see next section).

Various serological findings further confirm the presence of
immune system dysregulation and active inflammation in autism
- raised levels of proinflammatory cytokines have often been
observed in blood of patients with autism, with significant
increases of IFN-gamma, IL-6 and TNF-alpha. These results are
followed by findings of decreased peripheral lymphocyte numbers,
incomplete or partial T cell activation following stimulation,
decreased NK cells activity, dysregulated apoptosis
mechanisms, imbalances of serum immunoglobulin levels, increased
numbers of monocytes and abnormal T helper cell (Th1/Th2)
ratio, with a Th2 predominance, and without the compensatory
increase in the regulatory cytokine IL-10 [18762240,
16698940,
16360218].
It is of interest to note that, following increased levels of
TGF-beta1 in brain specimen as observed by Vargas et al, this
cytokine was found to be significantly lower in the blood of
adult patients with autism compared to controls [17030376,
18762342].

Another relevant observation is the elevation of cerebrospinal
fluid levels of TNF-alpha compared to its serum levels in subjects
with autism. The observed ratio of 53.7:1 is significantly higher
than the elevations reported for other pathological states for
which cerebrospinal fluid and serum tumor necrosis factor-alpha
levels have been simultaneously measured [17560496].

It has been suggested that prenatal viral infection might dysregulate
fetal immune system, resulting in viral tolerance in autism
[139400]
(see Infectious_Agents).
Studies showing altered T-cell subsets raised suspicions about
possibile autoimmune aspects of autism and several studies pointed
to association of the risk of autism to immune genes
located in the human leukocyte antigen (HLA) [16720216,
15694999].
The immune system uses the HLAs to differentiate self cells
and non-self cells and some of them are linked with higher risk
of autoimmune disorders. Animal studies show that behavioural
changes follow onset of autoimmune disease and can be reversed
through immune-suppressive treatments [8559794],
and although various autoantibodies to brain antigens have been
observed in autism, the results of those studies are often conflicting.
In addition to the inconsistent findings the question has been
raised as to whether those autoantibodies would be pathogenic
contributors or mere consequences of the the disorder. Of note
in this context is the finding pointing to possible association
of virus serology and brain autoantibodies in autism [9756729].
One study found that children with autism had a significantly
higher percent seropositivity of anti-nuclear antibodies. Anti-nuclear
antibody seropositivity was significantly higher in autistic
children with a family history of autoimmunity than those without
such history, and also had significant positive associations
with disease severity, mental retardation and electroencephalogram
abnormalities (19135624).
Autistic children were found to have significantly higher serum
anti-myelin-associated glycoprotein antibodies than healthy
children. Family history of autoimmunity in autistic children
was significantly higher than controls. Anti-myelin-associated
glycoprotein serum levels were significantly higher in autistic
children with than those without such history (19073846).

A study
looking at several markers of concomitant autoimmunity and immune
tolerance found highly elevated circulating IgA and IgG autoantibodies
to casein and gluten dietary proteins in autism sample compared
to controls. Circulating anti-measles, anti-mumps and anti-rubella
IgG were positive in only 50%, 73.3% and 53.3% of autistic children
previously immunised by MMR, as compared to 100% positivity
in the control group. Anti-cytomegalovirus CMV IgG was also
investigated and was positive in 43.3% of the autistic children
as compared to 7% in the control group (17974154).

One very interesting finding in recent times was the association
of genetic polymorphisms related to macrophage migration inhibitory
factor (MIF) in individuals with autism [18676531].
MIF is central in host immune reactions/viral clearance and
inflammatory responses. MIF favours viral neuroinvasion by compromising
the integrity of the blood-brain barrier (see Infectious
Agents re viral aetiology of autism). It is very closely
linked to MCP-1 (elevated manifold in autism) and other proinflammatory
chemokines, and its levels are inversely related to regulatory
cytokine IL-10 (low in autism). MIF also plays a central part
in gastrointestinal inflammation (see Gastrointestinal),
as well as cellular oxidative stress pathways - cysteine mediated
redox mechanisms (impaired in autism, see Oxidative
Stress). It is also appears to be directly involved in neuronal
function via at least one pathway, that of Angiotensin II. Levels
of MIF are often suppressed in fever. The expression levels
of MIF gene are regulated by calcium-dependent CREB.

In addition
to several mutations in genes belonging to major histocompatibility
complex (MHC II) region, related to immune
function and associated with autism, the involvement of CREB-mediated
events in regulation and expression of these genes
should be of utmost interest [16730065,
10458754]
(see Brain_Development).

Some aspects of calcium signalling in immunity and inflammation

Proinflammatory cytokines/chemokines are protein signals released
from a variety of cells in response to bacterial or viral infections
or agents that cause physical damage, for example oxalates,
in order to attract leukocytes to the site of inflammation.
CC chemokines induce the migration of monocytes and other cell
types such as NK cells and dendritic cells. One such example
is MCP-1, which induces monocytes to leave the bloodstream and
enter the surrounding tissue, becoming tissue macrophages.

Many inflammatory processes are mediated through activities
of LTCC and elevation in intracellular calcium level
is the central element in the activation of brain macrophages.
Several calcium channel blockers at therapeutic concentrations
were shown to modulate inflammatory processes. Benidipine for
example suppressed induction of MCP-1 and IL-8 [15364009].
In another study nifedipine inhibited TNF-alpha-induced reactive
oxygen species generation and subsequent MCP-1 [15921025,
15018305].
The activity of LTCC mediates the cyclic stretch-induced inflammatory
gene expression in airway smooth muscle and can be inhibited
by nifedipine, with the most responsive genes being the ones
encoding for COX-2, IL-6, and IL-8 [16339998,
8576944].
The induction of lymphocyte migration by IL-8, IL-1 alpha and
IL-1 beta also appears to be dependent on activities of VGCC
and can be inhibited by several calcium channel blockers [2686646].
TNF-alpha concentrations are shown to be suppressed in animals
pretreated with verapamil or diltiazem, and by nimodipine in
cultured monocytes [15661163].
On the other hand calcium entry blockers increase the regulatory
interleukin-10 production, shown to be consistently low in autism
[9110418].

Of special interest should be several recent observation of
reduced responsiveness to stimulation of both B and T lympocytes
following elevations of cytosolic free calcium [12805281]
(see Infectios_Agents).
Although LTCC are mainly expressed in excitable cells, recent
evidence points to the involvement of voltage-insensitive dihydropyridine
channels in calcium pathways of both B and T lymphocytes
[8631746,
9550376,
14981074].
Calcium influx through the plasma membrane of lymphocytes is
essential for their activation, proliferation, cytokine secretion
and apoptosis [16766050].
Calcium and CREB-mediated upregulation of chemokine receptors
mRNA has been observed in T lympocytes, whereby activation of
CXCR4 by viral proteins leads to increased viral infectivity
[11874984,
14563373].

Furthermore, differences in calcium mobilization are associated
with differentiation of naive CD4+ T cells
into Th1 and Th2 subsets. Because LTCC are induced during Th2
but not Th1 cell differentiation, it has been suggested that
LTCC blockers may be useful in the treatments of Th2-mediated
pathologies. One such agent, nicardipine, was able to inhibit
the Th2-mediated autoimmune glomerulonephritis induced by injecting
Brown Norway (BN) rats with heavy metals, but had no effect
on Th1-mediated experimental encephalomyelitis [15100258,
15777162,
14708347,
12721099].

Apoptosis plays an important role in the control of the immune
system, and its impairment may be associated with autoimmune
responses. Calcium entry through LTCC is thought to play a central
role in apoptosis-related processes, including apoptotic body
engulfment by dendritic cells.

Alterations in intracellular calcium levels have been implicated
in the pathophysiology of immune dysfunction. Elevation of calcium
levels play a central role in the regulation of executive functions
in activated microglia, the immunecompetent
cells in the brain, which have many similarities with macrophages
of peripheral tissues and also express a variety of ion channels.
Activation of microglia has been observed during infection,
inflammation, physical injury, trauma, ischemia, and in a number
of neurodegenerative disorders such as Alzheimer's disease,
Parkinson's disease, amyotrophic lateral sclerosis, multiple
sclerosis, and prion disease [12805281,
9914452].
It has been hypothesised that calcium influx via VGCC plays
a significant role in the development of neurological disability
and white matter damage in the animal experimental autoimmune
encephalomyelitis (EAE), an animal model of multiple sclerosis
(MS), and MS itself. Administration of calcium channel blockers
significantly ameliorated EAE in mice, with spinal cord samples
showing reduced inflammation and axonal pathology [15296830].
It should also be mentioned in this context that phosphodiesterase
(PDEIs) inhibitors have shown inhibiting effect on
cytokine production in lipopolysaccharide-stimulated mouse microglia.
PDEIs supressed the production of TNF-alpha, as well as IL-I
and IL-6. In contrast, the production of IL-10, was upregulated
by certain PDEIs, and it was concluded that these agenst may
be useful in treating inflammation in the CNS [10335522].
One of the possible explanations for the anti-inflammatory effect
of these agents is their regulatory effect on LTCC [7517800].

Mast cells are important for clearance of pathogens
and in wound healing, and play a central role in many forms
of allergies, like asthma and eczema. Upon stimulation mast
cells degranulate, releasing histamine, prostaglandin and many
cytokines. Because of their ability to release a wide range
of cytokines and other inflammatory mediators, they are thought
to have a central role in innate immunity and are implicated
in the pathology associated with inflammatory and autoimmune
disorders such as rheumatoid arthritis and multiple sclerosis.
The activation and release of both plasma cells and mast cell
contents is regulated by the influx of calcium, most probably
through Cav1.4 LTCC, which are highly expressed in those cells
[9357819].

Relative to autism it may be of interest to mention that a specific
mechanism has recently been suggested, whereas the crosstalk
between chemokine receptors and neuropeptide membrane receptors
serves as a bridge between the immune and nervous systems. Chemokine
receptors, a family of G protein-coupled receptors, are widely
expressed by cells of immune and nervous systems, including
neurons. The activation of several other receptors, such as
opioid, vasoactive intestinal peptide, or adenosine receptors,
often has inhibitory effects on chemokine receptors by a mechanism
termed heterologous desensitization, resulting in suppression
of immune responses. Conversely, activation of chemokine receptors
also induces desensitization of mu-opioid receptors (see Infectious_Agents
and Related_Receptors). In addition to that,
prior exposure of neuronal cells to chemokine treatment enhances
the sensitivity of transient receptor potential vanilloid 1
(TRPV1) calcium channel, which is critical for sensing of pain
[16204635]
(see Sensory/Motor).

Apart from playing an important role in functioning of lymphocytes,
chemokines can potentially influence neuronal signaling
through the modulation of neuronal calcium currents
(see chapter on Chemokine
Receptors and Calcium Homeostasis in the Brain).
Following interaction with their specific ligands, chemokine
receptors trigger a flux in intracellular calcium ions in neurons.
This process is sensitive to treatment with pertussis toxin,
indicating the involvement of G-protein couple receptors in
these mechanisms (see above) [11880151].

Similarly, some chemokines are able to induce a rapid and transient
rise in cytosolic free calcium in in either type of T-cell via
activation of their ligands [7926371].
It may be of interest to mention here a novel model of reversible
inflammatory encephalopathy that is found to be dependent on
both genetic and environmental factors. In a study that investigated
the pertussis toxin-induced encephalopathy in mice, it was shown
that transgenic mice that overexpress MCP-1 manifest transient,
severe encephalopathy with high mortality after injections of
pertusis toxin, whereas this disorder was significantly milder
in mice lacking T-cells. Disruption of CC chemokine receptor
2 (CCR2) abolished both CNS inflammation and encephalopathy
in this case [12486156]
(see Bacterial lypopolysaccharides)

Several viral proteins that are able to directly interfere with
human chemokine receptors have been identified. In the case
of the most widely-studied HIV-1, two of its proteins mimick
chemokine-mediated calcium signaling and evoke calcium influx
through LTCC via activation of several chemokine receptors.
One of the chemokine receptors involved, CXCR4, is known for
its involvement in immune dysfunction thought to be due to dysregulation
of the receptor leading to to enhanced calcium signaling [17169327]
(see Infectious_Agents).
Through this mechanism it is thought to be able to modulate
the migratory and secretory responses of microglia or cause
raises in calcium levels in neurons resulting in neuronal injury
[10858625,
16553776].
Of particular relevance to autism could be the observed ability
of retroviruses to induce neuronal damage even in the absence
of lympocyte infiltration, by upregulation of chemokine MCP-1
and subsequent overactivation of it receptor CCR2 [15163738].

Human herpesvirus 6 (HHV-6) encodes highly effective and adaptive
chemokine receptor agonists, which will attract CCR2 expressing
cells, including macrophages and monocytes. At the same time
these proteins enable viral interference in calcium signalling
pathways and thus influence many downstream cellular processes.
HHV-6 encoded U83A gene is able to induce significant
calcium mobilization in cells expressing chemokine
receptors CCR1, CCR4, CCR6, or CCR8, with high afinity to both
CCR1 and CCR5 on monocytic/macrophage cells. It was concluded
that this newly uncovered mechanism could have wider implications
for neuroinflammatory diseases such as multiple sclerosis, where
both cells bearing CCR1/CCR5 plus their ligands, as well as
HHV-6A, have been suggested to play etiological role [16332987,
16365449].
(also see BBB)

Virus-induced immune suppression has been documented with numerous
viruses including other members of the herpes virus family,
which are able to lower antigen presenting ability of myocites
via impaired differentiation of monocytes to dendritic cells,
impaired migration of dendritic cells [15914842],
loss of up regulation of MHC protein (both Class I and Class II)
expression, and loss of up regulation of CD4 cells and CD8 cytotoxic
cells, altered cytokine production and activity, altered T cell
recognition, and loss of NK function with lack of viral killing
[16647570,
1315587,
9087413].
CD4 and CD8 cytotoxic cells and NK cells are affected either
directly or indirectly by HHV-6 [9227865,
1348547].
The impairment of immune function by herpes and other viruses
may lead to reactivation or emergence of other opportunistic
micro-organisms that persist chronically or in a latent state,
for example Epstein Bar Virus (EBV), chlamydia, mycoplasma,
Borrelia burgdorferi, and babesia, among others. Worth mentioning
in this context is the finding that a large subset of subjects
with autism shows evidence of various bacterial and/or viral
infections not present in age-matched controls (see Infectious_Agents).

It is suggested that one of the mechanisms through which herpesviruses
exerts its immunosuppressive effects is via dysregulation of calcium
homeostasis in immune cells [14568989].
Another possible example of this mechanism is the effect of
HIV-1 viral protein Tat, which is known to inhibit functioning
of dendritic cells and NK cells by dysregulating the functioning
of LTCC [9516412,
9743356].
In cultured human primary monocytes Tat induces a calcium signal
by mobilizing calcium from extracellular stores. This effect
is again mediated via calcium influx through LTCC [15661163].
Another viral protein, belonging to Hepatitic C, is known to
alter expression of genes involved in cell adhesion and motility,
calcium homeostasis, lipid transport and metabolism, as well
as genes regulating immune and inflammatory responses [15349911].

Apart from viruses various other agents are thought to have
immunosupressing properties and can act as stressors, negatively
influencing proliferative responses of cells. Various toxic
chemicals have been shown to induce immune dysregulation
via their effects on calcium homeostasis. Cadmium-induced increases
in intercellular calcium levels in macrophage cells lead to
cellular dysfunction and cell injury, including growth arrest,
mitochondrial activity impairment, and necrosis [15254339].
Exposure of cultured murine macrophages to low concentrations
of beryllium also induced increases in intracellular calcium
and subsequent DNA synthesis. This effect could be reversed
by removing extracellular calcium or applying verapamil, indicating
the involvement of plasma membrane calcium channels [10380900].
Tin appears to exert similar effects on immune cells via calcium
mobilisation [9138773].

Mercurial compounds are particularly toxic
to the human immune system. Chronic low-level methylmercury
(MeHg) exposure may cause immune disfunction by increasing oxidative
stress, affecting transmembrane calcium signaling and splenic
cellularity [9653674].
In cultured murine T and B lymphocytes mercury incudes cytotoxicity
via several mechanism that involve disturbances in calcium homeostasis
and inflammatory cytokine gene expression [12849721].
Adverse affect of methylmercury on the developing immune system
through placental and lactational transfer has been demonstrated
in rats [1949074].
Intracellular levels of free calcium play an essential role
in neutrophil activation, and methylmercury increases those
levels in the mouse peritoneal neutrophils via LTCC [12062935].
Exposure of human cultured lymphocytes to methyl mercuric chloride
(MeHgCl) results in a sharp decrease in the mitochondrial transmembrane
potential in lymphocytes, followed by an increase in oxidative
stress [12215206].
Dendritic cells, especially immature dendritic cells, appear
to be very sensitive to the effects of organic compound ethylmercurithiosalicylate
(thimerosal, primarily present in the tissues as ethylmercury) on
calcium homeostasis. Uncoupling of ATP-mediated calcium signaling
and dysregulated interleukin-6 secretion has been observed in
murine cells following application of relatively small levels
thimerosal, with one mechanism involving the uncoupling of ryanodine
receptor RyR1 calcium channel complex [16835063].
It should be noted here that in many cells types ryanodine receptors
of endoplasmic/sarcoplasmic reticulum form a functionally and
structurally connected signalling unit with LTCC on the plasma
membrane, and this signaling interaction between LTCC and RyRs
is often bi-directional, in that the LTCC trigger the release
of intracellular calcium by promoting the opening of nearby
RyR (orthograde coupling) while at the same time the activities
and function of LTCC can regulated by its interaction with the
RyR (retrograde coupling) [11861208].

(see also chapter: Genetic
Polymorphisms as Predictors of Inflammatory Outcomes and Chemokine
Receptors as Treatment Targets)